1. Field of the Invention
The present invention relates to recombinant C-terminal xcex1-amidated enzymes of Xenopus laevis origin and precursors thereof, DNAs coding for these poly-peptides, plasmids containing the DNA, host cells transformed with the plasmid, a process for the production of the enzyme or precursor thereof using the transformed cells, and a process for the production of a C-terminal xcex1-amidated peptide or protein using the enzyme.
2. Related Art
It is generally known that, in eukaryotic cells, some kinds of peptides or proteins are, after translation from a messenger RNA (mRNA), modified by an intracelular enzyme to mature to a natural-type peptide or protein (post-translational modification). But, since prokaryotic hosts such as E. coli, which are widely used to produce peptides or proteins of eukaryote origin, cannot carry out a post-translational modification of an expressed peptide or protein, it is sometimes difficult to directly produce a eukaryotic peptide or protein by a recombinant DNA technique using prokaryotic host cells.
One of this post-translational modification characteristic of eukaryotic cells of peptides or proteins is a modification reaction wherein an xcex1-position of a carboxy terminal (C-terminal) of a peptide or protein is amidated, i.e., xe2x80x94COOH is converted to xe2x80x94CONH2, and it is known that many physiologically active peptides or proteins have been subjected to such modification. For example, as C-terminal xcex1-amidated peptides (SEQ ID NOS:1 and 2), TRH(pGlu-His-Pro-NH2) and 
have been isolated, and a partial structure of precursors of these peptide determined from an analysis of the CDNA thereof. A general biosynthesis mechanism of such amidated peptides is understood to be that in which RNA is translated to a precursor of an amidated peptide, which is then amidated at the xcex1-position of the C-terminal thereof by a C-terminal xcex1-amidating enzyme. Note, in the above-mentioned reaction, the precursor of the C-terminal xcex1-amidated peptide as a substrate for a C-terminal xcex1-amidating enzyme is a peptide or protein represented by a general formula Rxe2x80x94X-Gly, wherein R represents an amino acid sequence of the N-terminal side of the peptide or protein, X represents an amino acid residue which is to be xcex1-amidated at the C-terminal thereof, and Gly represents a glycine residue.
It is known that, in some cases, the above-mentioned modification of peptide or protein is essential to the physiological activity thereof. For example, a conversion of the proline amide residue at the C-terminal of natural-type human calcitonin to proline residue decreases the physiological activity thereof to {fraction (1/1,600)} of the original activity.
Because of the importance of clarifying the mechanism of a-amide formation in tissues, and the promising usefulness of the enzyme for the production of C-terminal xcex1-amidated peptides using, for example, recombinant DNA techniques, many attempts to purify the enzyme have been made but the enzyme has not so far been obtained in a pure state. In porcine pituitary, Bradburg, A. F. et al, Nature 298, 686-688, 1982, first characterized the xcex1-amidating activity converting a synthetic substrate D-Tyr-Val-Gly to D-Tyr-Val-NH2, and demonstrated that the C-terminal glycine in the substrate serves as a nitrogen-donor for xcex1-amidation. Eipper et al, Proc. Natl. Acad. Sci. US, 80, 5144-5148, 1983, reported that the xcex1-amidated enzyme derived from the pituitary gland requires copper cation and ascorbate for its activity. Husain, I. et al, FEBS Lett., 152 227-281, 1983; and Kizer, J. S. et al, Proc. Natl. Acad. Sci. US, 81, 3228-3232, 1984, also reported a C-terminal xcex1-amidating enzyme, but did not report a purified enzyme. Recently, Murthy A. S. N. et al, J. Biol. Chem., 261, 1815-1822, 1986, partially purified a C-terminal xcex1-amidating enzyme from the pituitary gland of cattle, and showed that several types of enzymes having different molecular weights and electric charges are present. Nevertheless, no type of enzyme has been homogeneously purified.
Recently, Mizuno et al. succeeded in isolating a C-terminal xcex1-amidating enzyme in a homogeneous and pure form from a skin of Xenopus laevis; see Mizuno, K et al, Biochem. Biophys. Res. Commun. 137, 984-991, 1988, and Japanese Patent Application No. 61-131089.
Nevertheless, the amount of the C-terminal xcex1-amidating enzyme isolated from a skin of Xenopus laevis is limited, and not sufficient for use in the industrial production of C-terminal xcex1-amidated peptides or proteins.
Accordingly, the present invention provides a recombinant DNA technique for the production of a C-terminal xcex1-amidating enzyme and the large amount of thus-produced enzyme, as well as the use of the enzyme for the production of C-terminal xcex1-amidated peptides or protein.
More specifically, the present invention provides a C-terminal xcex1-amidated enzyme of Xenopus laevis and precursors thereof produced by a recombinant DNA technique.
The present invention also provides a DNA coding for a C-terminal xcex1-amidating enzyme of Xenopus laevis or precursor thereof.
Further, the present invention provides a plasmid containing a DNA coding for a C-terminal xcex1-amidating enzyme of Xenopus laevis or precursor thereof.
Moreover, the present invention provides host organisms transformed with a plasmid containing the DNA coding for a C-terminal xcex1-amidated enzyme of Xenopus laevis or precursor thereof.
Still further the present invention provides a process for the production of a C-terminal xcex1-amidating enzyme of Xenopus laevis and precursor thereof comprising the steps of, culturing a host organism transformed with a plasmid containing a DNA coding for the enzyme or precursor thereof to produce the enzyme or precursor thereof, and recovering the enzyme or precursor thereof.
Also, the present invention provides a process for the production of a C-terminal xcex1-amidated peptide or protein characterized by reacting the above-mentioned enzyme with a peptide or protein having a glycine residue at the C-terminal thereof.